There are many medical uses at the present time for antigenic fractions: serotherapies of all kinds, serodiagnosis, detection of pathogenic germs in the widest range of pathological substances, epidemiological investigations and in particular vaccinations. As is known, vaccination is often the only weapon and the only prophylaxis which is available against many illnesses, hence the importance afforded by researchers to the development of processes for producing antigens which have a high level of antigenicity and a high degree of specificity. Research has first been directed towards producing an immunization effect with regard to bacterial infections, in particular enteric infections, which are induced for example by bacteria of the group of Salmonella and more particularly Salmonella thyphimurium, by means of a vaccine comprising entire killed bacteria. However, the use of such vaccines is not entirely without danger because of their toxicity due to the substantial endotoxin content in the cellular membrane of said bacteria, which toxicity is the cause of allergic reactions which are sometimes very serious. Attempts have therefore been made to prepare vaccines which comprise purified fractions extracted from bacterial cultures. Thus, there have been proposed vaccines which are produced from surface antigenic fractions extracted from Brucella melitensis and Brucella abortus, which essentially comprise a lipopolysaccharide-protein complex (cf DIAZ, JONES, LEONG and WILSON, Journal of Bacteriology, October 1968, volume 96, No. 4, pages 893-901, `Surface antigens of smooth Brucellae`). It should be noted that the writers of the above-quoted Article report that, if the active constituent of the antigenic fraction is the above-mentioned lipopolysaccharide-protein complex, the antigenic fraction nonetheless also contains proteinic components associated with internal Brucellae antigens, and a polysaccharide-protein complex which is without any lipid, of which it is said that it would apparently not play an important part in agglutination reactions because its antibodies could be separated by absorption without affecting the rate of agglutination. Likewise, there have been proposed vaccines which are polyvalent against Pseudomonas aeruginosa, which are produced from surface antigens having a high level of immunogenicity and which essentially comprise a highly immunogenic lipopolysaccharide-protein complex which is without toxic constituents, extracted from cultures of bacteria on a special medium.
Another proposed solution for eliminating the toxicity due to the endotoxin of the membranes of the cells of the bacteria is represented by acellular vaccines which, as the source of antigens, use the excretion products (or `slime`) of bacterial cultures, in particular cultures of Pseudomonas aeruginosa, which have previously been freed, by a washing operation, of the toxic products which are also contained in the slime, in particular labile excretion products and impurities from the culture medium (see French Pat. No. 2 290 219 of Nov. 5th 1974 to INSTITUT PASTEUR and P BERCHE, M VERON and R TINELLI, Ann. Microbiol. (Institut Pasteur) 1976, 127A, pages 247-259).
However, these various attempts at purifying bacterial cultures have not achieved their aim in that on the one hand they have not made it possible effectively to identify the antigenic constituent capable of giving the desired immunization effect and on the other hand they have not made it possible to produce vaccines which are effectively without toxicity. Research has therefore been continued in these two directions. Thus, there has been proposed an antigenic fraction which is extracted by suitable processes from cultures of pathogenic germs, in particular cultures of Salmonella thyphimurium, Malleomyces mallei, Pseudomonas mallei, and Vibrio cholerae, which would not have the allergic reactions of the previously proposed vaccines; this antigenic fraction which was identified as being a lipido-proteinic fraction is characterized in that it corresponds to the first peak on the diagram of absorption in ultra-violet at 280 nm, and that, when opposed to an immune-serum produced in an animal from the same culture, it gives the band (or the pair of bands) which is closest to the antigen well in the test of diffusion in a gelose medium and in the immunoelectrophoretic analysis test (see French Pat. No. 73 03734 of 2nd February 1973 to ANVAR, inventor: A DODIN). It has been found however that none of the attempts at immunization by the proposed purified antigenic fractions was satisfactory, because those fractions all contain a lipidic portion to which it was possible to attribute toxicity (see Lu DERITZ, GALANOS, LEHMANN, NURMINEN, RIETSCHEL, ROSENFELDER, SIMON and WESTPHAL, 1973, `Lipid A: Chemical structure and biological activity` in `Bacterial lipopolysaccharides`, edited by KALL and WOLFF, the University of Chicago Press, Chicago). Although experiments were carried out in order to obtain immumization in mice with respect to Pseudomonas aeruginosa, by capsular polysaccharides extracted from Pseudomonas aeruginosa slime (see PIER, SIDBERRY & SADOFF, INFECTION and IMMUNITY, December 1978, volume 22, No. 3, pages 908-918 and PIER et alia, loc. cit. pages 919-925), it is nonetheless admitted that it is not possible to prepare vaccines from polysaccharides, whether they are extracted from the excretion products of bacterial cultures or produced by cutting the lipidic portion of the lipopolysaccharides (LPS) molecule, considering that such vaccines have a very low level of immunogenicity which is because the polysaccharides, both those which are capsular and those which are produced from the slime, if being responsible for antigenic specificity, are devoid of immunogenicity.
It has also been proposed that mice should be immunized with regard to Bordetella pertussis, by means of an oligopeptide isolated from that bacterium, from a group of ribonucleoproteins of low molecular weight (see WILHELM & ROMER, zbl. Bakt. Hyg., I Abt., Orig. B 166, pages 264-271 (1978)), immunisation by peptidic complexes of organisms containing de-oxyribonucleic acids, which complexes are presented as having specific antigenic properties with regard to the organism from which they are isolated, also being proposed by French Pat. No. 2 387 991 of 19th April 1978 to R. and Z. VERMO GENSVERWALTUNGSGESELLSCHAFT GmbH.
However, prior to the latter publications, the proteins associated with the endotoxin present in the cellular membrane of the bacteria were considered as ,dditives suited to stimulating the production of antibodies and not as having an antigenic activity in themselves (see AMSTEDT & LINDHOLM, Immunology, 33, pages 629-633 (1977)). Likewise, a certain number of patents have been filed for vaccines in which the polysaccharides and/or the glycopeptides are included as immunity and stabilization additives associated with ARN or the ribosomes extracted from pathogenic germs.
Another direction in which research has been carried on consisted of preparing a synthetic polysaccharide-protein conjugation product which can be used as an active immunogen with regard to different pathogenic germs (in particular Pneumococci, Salmonella), starting from the consideration that the polysaccharide portion of the membrane lipopolysaccharide (LPS) of the bacteria enjoys antigenic specificity whose importance is primordial with regard to the immunogenicity of the vaccine, and that, if that polysaccharide could be linked as haptene constituting an excellent antigenic determinant, to a carrier formed by an immunogenic protein without toxicity, the result which would be achieved is an immunogenic agent for the preparation of specific vaccines (see PAUL, KATZ and BENACERAF, The Journal of Immunology, volume 107, No. 3, September 1971, pages 658-688; EKBORG et alia, Immunochemistry (1977), volume 14, pages 153-157; SVENSON and LINDBERG, FEMS Microbiology Letters 1 (1977), pages 145-148; LINDBERG et alia, INFECTION & IMMUNITY, volume 10, No. 3, September 1974, pages 541-545; SVENSON and LINDBERG, The Journal of Immunology, volume 120, No. 5, May 1978, pages 1750-1757; ZOPF et alia, Archives of Biochemistry and Biophysics, volume 185, No. 1 (1978), pages 61-71; SVENUNGSSON and LINDBERG, Acta Path. Microbiol. Scand. Sect. B,86, pages 35-40 (1978)). However, the comparative tests carried out by the different researchers have shown that, if such synthetic conjugation products may indeed have immunogenic activity and the absence of toxicity which was expected thereof, their immunogenic activity is nonetheless very much lower than that of the vaccines produced from entire dead bacteria.